Evaluation of pesticide residue in rice, wheat and pulses of Bidar district Karnataka, India

Issues in Biological Sciences and Pharmaceutical Research Vol.3(9),pp.100-106,September 2015 Available online at http://www.journalissues.org/ibspr/ http://dx.doi.org/10.15739/ibspr.019 Copyright 2015 Author(s) retain the copyright of this article ISSN 2350-1588 Original Research Article Evaluation of pesticide residue in rice, wheat and pulses of Bidar district Karnataka, India Received 10 August, 2015, Revised 26 August, 2015 Accepted 30 August, 2015 Published 11 September, 2015 1 Jagadish G.K., 2 Jaylakshmi S.K and 1 *Sreeramulu K 1 Department of Biochemistry, gulbarga University, gulbarga- 585106,India 2 College of Agriculture (University of Agricultural Sciences-Raichur), Aland Road, Gulbarga-585101,India. *Corresponding Author Email: ksramu@rediffmail.com Tel.: +91-8472-263289, Fax : +91-8472-245632 Pesticide residues in rice, wheat and pulses of Bidar district of north Karnataka were evaluated in this study. Organophosphates (OP), Organochlorines (OC), Synthetic pyrethroids (SP) and carbamate residues were monitored. A total of 250 samples from different markets of Bidar district, Karnataka were analysed by gas chromatography (GC) and high performance liquid chromatography (HPLC). Recovery studies carried out for all 4 groups of pesticides were within the acceptable range of 70-120% before the analysis of field samples. Sample extraction and partitioning was carried out by multi residue method. Among the samples analysed, 80 samples were found to be contaminated with different groups of pesticides. Pesticide residues were found to be above the Maximum Residue Limit (MRL) in 22 samples and below MRL in 58 samples. More numbers of pesticides were detected in wheat followed by rice and pulses. In pulses, red gram was the major commodity contaminated with multiple pesticide residues followed by black gram and green gram. OP pesticides were dominated OC and SP in all the studied commodities. Most commonly detected OPs were chlorpyrifos, parathion, profenofos, ethion and triazofos. Key words: Pesticides, GC, HPLC, pulses, rice and wheat INTRODUCTION Karnataka is one among the major states in production of pulses, rice, wheat, sorghum, fruits and vegetables. The north regions of Karnataka (Bidar) have semi-arid dry conditions with temperatures ranging from 26-40 o C in summer and the agriculture is mainly dependent on rain. The major crops grown in these areas are red gram, green gram, black gram; wheat and rice; fruits like mango and banana. India is an agriculture based country with second largest producer of vegetables after china, and access for 13.4% of the world production. The growing demand for food and feed is due to consistent increase in population and the use of chemical pesticides has been increased to obtain more yield and protection of crop from pests and diseases. Through which the crop protection has been increased to 100% but the cropping area has increased marginally 20%. But the indiscriminate usage of pesticide leads to accumulation of pesticide residues in food chain, aquatic system and soil (Jayashree and Vasudevan, 2007). The non-degradability of pesticide residues causes resistance of pests to the chemicals indicating 50-70% of contamination with insecticide residues. Public concern towards pesticide residues has risen over the few decades to identify the point where it has become a significant food safety issue (Column et al., 1999). The determination of pesticide residues in food has become an essential requirement for consumer, producers and authorities responsible for food quality control (Aguilear et al., 2003). Environmental pollution is one of the serious predicaments of the modern world (Hela et al., 2005). During the last decade, significant increase in environmental pollutants and lack of precautionary measures or observance of the environmental regulations has become a global problem (Bondarenko et al., 2004; Hela et al., 2005; Abdel-Halim et al., 2006; Wilsont and Foos, 2006). Pesticides applied to food crops in the field can leave potentially harmful residues; OC in particular is persistent in foodstuffs for longer periods. If crops are sprayed on to

Issues Biol. Sci. Pharma. Res. 101 harvest without an appropriate waiting period, even OP can persist in food (Bull, 1992). Pesticide residues in foods are a growing source of concern for the general population (Torres et al. 1996). A substantial body of laboratory and epidemiological evidence suggests that certain pesticides are associated with carcinogenesis, immunotoxicity, neurotoxicity, behavioural impairment, reproductive dysfunction, endocrine disruption, developmental disabilities and respiratory diseases, such as asthama (Solomon et al., 2000). Cereals and pulses form a large proportion of the global diet and control of pests during storage is a bigger problem. Storage pests can be controlled by the use of insecticides as residual protectants. This grain treatment method can be easily adaptable to all types and sizes of storage with minimum expense. This post harvest application of pesticides leads to the persistence of pesticides in food grains and may cause health hazards (Webley, 1994). The short as well as long term impacts of the use of pesticides on biological systems are being evaluated continuously in an effort to minimize the hazards. A wide spread use pesticides, their toxic residues have been reported in various environmental matrices by the researchers (Kumari et al., 2002; Kumari et al., 2003; Singh et al.,2004). The presence of pesticide residues in rice, wheat and pulses can be a significant route to human exposure and most of (OC) pesticides have been banned because they are highly persistent insecticides, and their residues still appear as pollutants in food and environment (European Community, 1990). Organochlorine pesticides are characterized by high lipid solubility and high persistence and hence they tend to accumulate in fatty tissues. To ensure the safety of food for consumers, numerous legislations such as the European Council (EC) directives have established MRLs for pesticides in food. No reports have yet been published about OC, OP, SP and carbamate pesticides in pulses, wheat and rice from North Karnataka. In the present study, the monitoring of OC, OP & SP in cereals and pulses, which are undesirable substances in food according to the Indian Community directives, have been investigated. MATERIAL AND METHODS Primary secondary amine (PSA) was procured from Agilent Technologies, Pvt. Ltd, India. Reference standards of OC, OP, SP and Carbamates were procured from Dr. Ehrenstorfer (Augsburg, Germany). All other chemicals used were of analytical grade. Sample collection A total of 250 samples of cereals namely rice, wheat and pulses (red gram, black gram and green gram) cultivated during the kharif season of the year 2014 were collected from different farmers fields, whole sale market and retail markets of Bidar district, which includes 5 taluk s namely Bhalki, Aurad, Basvakalyan, Humnabad and Bidar. From each taluk, 10 samples of rice, wheat, red gram, black gram and green gram were collected in air tight polyethylene zip cover bags and stored in refrigerator until complete analysis. Approximately 1-2 kg of each sample was collected. Sample extraction and purification was completed within 24 hours of collection. Preparation of standard solutions Stock solutions of OP mixture, OC mixture and SP mixture (500 µg ml -1 ) were prepared in hexane: acetone (9:1). Stock solution of carbamates (500 µg ml -1 ) was prepared in HPLC grade acetonitrile. OP mixture contains phorate, dichlorovos, methyl parathion, dimethoate, fenitrothion, profenofos, malathion, chlorpyrifos, quinalfos, ethion, and triazofos. Mixture of OC contains alpha-hch, gamma-hch, alpha-endosulfan, endosulfan sulphate, aldrin and p.p -DDT. SP mixture contains bifenthrin, fenpropathrin, cyhalothrinlambda, cypermethrin and deltamethrin. Working standard solutions were prepared by further dilutions of the stock solutions. Extraction and Purification Cereal and pulse samples (1-2 kg) were homogenized in a warring blender. To the flask containing 25 g of a powdered sample, 100 ml mixture of water and acetone (35:65 v/v) was added and kept for shaking for about 30 minutes in a mechanical shaker and filtered under vacuum through a Buchner funnel. The extract was transferred to a flat bottom flask and concentrated to near dryness in a rotary evaporator. The aqueous extract was partitioned using 50 ml mixture of hexane: dichloromethane (1:1). The process was repeated twice. The organic fraction obtained after the partitioning was transferred to a 250 ml flat bottomed flask filtered through sodium sulphate to absorb the residual moisture. The organic portion was concentrated up to dryness in a rotary evaporator and redissolved using 10 ml n-hexane: acetone (1: 1). Final volume of 10 ml sample was taken in a 25 ml centrifuge tube and 250 mg primary secondary amine (PSA) and 1 g magnesium sulphate was added to it. The contents of the tubes were mixed in a vortex mixer and centrifuged at 10000 rpm for 10 mins. 3 ml of this sample was evaporated and re-dissolved in HPLC grade acetonitrile for analysis in HPLC. The samples were transferred to 1.5 ml glass vials for analysis by GC and HPLC. Chromatographic analysis Analysis of OP, OC and SP residues was performed using a Bruker gas chromatograph (GC), Model 450-GC. Electron capture detector (ECD) was used for the analysis of OC and SP and nitrogen phosphorous detector (NPD) was used for the analysis of OP. A capillary column DB-5MS (30m 0.25mm, 0.25µm film thickness; stationary phase, 5% phenyl / 95% dimethylarylene siloxane) was used for the

Jagadish et al. 102 Figure 1: GC chromatogram of selected OP mix 1ppm 1.Dichlorovos, 2.Phorate, 3.Dimethoate, 4.Methylparathion, 5.Fenitrothion, 6.Malathion, 7.Chloropyriphos, 8.Quinolphos, 9.Ethion, 10.Triazophos analysis. The injector was kept at split less mode. Column oven was initially maintained at 80 C with a hold time of 1 min, then increased at 15 C min 1 to 150 C with a hold time of 5 min and then increased at 4 C min 1 to 250 C and further increased at 10 C min 1 to 280 C with a hold time of 15 min. The temperatures of the injector and detector were maintained at 280 C and 300 C, respectively. One microliter of samples was injected with the auto-sampler. Ultra high pure nitrogen was used as the carrier gas at a flow rate of 1 ml min 1. At the above mentioned conditions the analysis of pesticide residues was performed. Analysis of carbamate residues in cereals and pulses were carried out by HPLC (Shimadzu, LC 2010 CHT) with a PDA detector. The column used was Phenomenex Luna C- 18, 100A, 250 4, 60mm. The mobile phase composition used was acetonitrile: water (50:50, v/v) at a flow rate of 1.0 ml min -1 and an injection volume of 20µL was used. Method validation The multi residue method (AOAC-2000) used for the analysis of all 4 groups of pesticides (OC, SP, OP and carbamates) was performed according to SANCO guidelines (SANCO, 2013). The recovery experiments were carried out to determine the accuracy of the method by using the above mentioned procedures. The limit of detection (LOD), the lowest amount of analyte that can be detected with a signal to noise ratio of 3 was determined by analysing lower concentrations of pesticides. Limit of quantification (LOQ), the lowest amount of analyte that can be detected at a signal to noise ratio of 10 was determined by analyzing the lower concentration standards prepared in the blank extracts of rice, wheat and pulses. Linearity curve was drawn by analysing standards in the range of 0.01-1 µg ml - 1. RESULTS AND DISCUSSION The method used was efficient to extract and analyse all the 4 groups of pesticides. The recoveries obtained were within the acceptable range of 70-120%. The LOD of OC and SP was 0.01 µg ml -1 ; LOD of OP was 0.02 µg ml -1. Carbamates were determined at the LOD of 0.1 µg ml -1 (Figure 3). The LOQ of OC, SP and OP in cereals and pulses was 0.05 mg kg - 1 ; LOQ of carbamates in cereals and pulses was 0.5 mg kg -1. The method was linear for all the pesticides with a correlation coefficient >0.99. The GC chromatogram of OP standard mixture is given in Figure 1. Pesticide residues in cereals Rabbi and kharif are two major cultivation patterns in Bidar mainly depend on rain. Major crops in this area are pulses, wheat, and nuts; while minor are sugarcane, oil seeds and vegetables. Wheat is the optimum crop for the farmer in this area. Out of 50 wheat samples analysed from different sources, 24 samples were contaminated with pesticide residue in which 8 are above MRL and 16 samples are below MRL (Table 2). In wheat and rice, the major groups of pesticides found were OP dominated most than SP followed by OC; carbamates were not found in any of the samples analyzed (Table 1). Among OP, chloropyrifos is the most abundant pesticide residue in wheat and rice followed

Issues Biol. Sci. Pharma. Res. 103 Table I. Detection of pesticides in different commodities collected from different places of Bidar District of Karnataka state, India Commodity Sample collection point Rice Wheat Red gram Black gram Samples collected from farmers field Sample Pesticide analysed residue detected Samples collected from wholesale market Sample Pesticide analysed residue detected Samples collected from retailers shop Sample Pesticide analysed residue detected 20 1 20 2 10 1 20 1 20 3 10 2 20 1 20 2 10 1 20 0 20 2 10 1 Green gram 20 2 20 1 10 1 Figure 2: Number of Pesticide contamination in different commodities and type of pesticides detected. by profenofos, malathion, whereas, triazofos concentration is very negligible. Synthetic pyrithroids (cypermethrin and cyhalothrin-lambda) are found in the wheat. However, out of 50 rice samples 18 are found to be contaminated as shown in Figure 2, in which 4 samples are above the MRL and rest are below MRL. Similar results in cereals were also reported in Tomer (2013). Pesticide residues in pulses Major cultivation crops in pulses are red gram, black gram and green gram. Red gram is found to be contaminated with more number of pesticides followed by black gram and green gram. Table 2 shows out of 50 samples of each pulses, 17 red gram, 9 black gram and 12 green gram are

Jagadish et al. 104 Table 2. Pesticide contaminated more than MRL and MRL of detected pesticide Commodity No. of samples contaminated with pesticide residues No. of samples with pesticides more than MRL Pesticides detected MRL of pesticide (EU MRL) (mg/kg) Rice 18 04 Chloripyriphos 0.05 Triazofos 0.02 Bifenthri 0.01 Fenpropathrin Malathion 0.02 aldrin 0.01 Wheat 24 06 Chlorpyrifos 0.05 Triazofos 0.02 Malathion Aldrin 0.01 Cypermethrin 2 Dichlorovos Cyhalothrin-L 0.05 Red gram 17 04 Chlorpyrifos 0.05 Triazofos 0.01 Malathion Cypermethrin 0.05 Black gram 09 03 Chlorpyrifos 0.05 Triazofos 0.01 Cypermethrin 0.05 Green gram 12 04 Chloripyriphos 0.05 Triazofos 0.01 Malathion Cypermethrin 0.05 contaminated with pesticide residues. The pesticides found above MRL in 4 red gram, 3 black gram and 4 green gram samples. Multiple pesticides are detected in cereals and pulses may be due to cocktails of various pesticides to increase the potency of the compounds as reported by Danso et al. (2002) and Ntow et al. (2006). The most commonly detected pesticide in cereals and pulses is chloropyrifos which has effect on health and safety of mammals has been assessed in numerous studies by Johnson et al. (1998); Clegg et al. (1999). Poisoning with this compound can affect the central nervous system, cardiovascular and respiratory system (Nolan et al., 1984). Tomer (2013) elucidated the percent contamination of pesticides in the pulses. An attempt has been made to study the monitoring and evaluation of pesticide residues in grown food grains of Bidar area. This study suggests that minimize the indiscriminate, non-recommended usage of pesticides on food grains. Study of pesticide residue in holoestic pattern is prime importance to implement the act in this region. Table 2 depicts out of 50 samples of each commodity 20 samples from whole sale 20 from farmers field and 10 from retail shop was collected. Most of the wholesale and retail samples are contaminated with the OP, OC and SP pesticide residues followed by farmer field s samples, there was no detection of carbamates in any of the pulses samples. Study concludes that food grains and pulses are cross contaminated in storage may be by post-harvest application of pesticide. Detection of these pesticide residues shows that the serious concern for the food grains and human health. Measures to be taken for the monitoring and evaluation of pesticide residue at consumer level. Acknowledgement One of the author Jagadish G.K. would like to thanks

Issues Biol. Sci. Pharma. Res. 105 Figure 3: HPLC chromatogram of methomyl (a) and mixture of carboryl and carbofuran (b) agriculture research station Gulbarga for providing CRM s for this study. REFERENCES Abdel-Halim KY, Salama AK, El-Khateeb EN, Bakry NM (2006). OP pollutants (OPP) in aquatic environment at Damietta Governorate, Egypt: Implications for monitoring and biomarker responses. Chemosphere., 63: 1491-1498. Aguilear A, Brotons M, Roclriguez M, Valverele A (2003). Supercritical fluid extraction of pesticides from a table-ready food composite of plant origin (Gazpacho). J. Agri. Food chain., 51: 5616-5621. Bondarenko S, Gan J, Haver DL, Kabashima JN (2004). Persistence of selected organophosphate and carbamate insecticides in waters from a coastal watershed. Environ. Bull D (1992). A growing problem: Pesticides and the Third World poor. Oxford: OXFAM Clegg DJ, Gemert MV (1999). Determination of the reference dose for Chloropyrifos; Proceedings of an experimental panel. J.Toxicol. Environ. Health Part B. Crit: Rev., 2: 211-214. Column A, Cardenas S, Gallego M, Valcarcal M (1999). Semiautomatic method for the screening and determination of 23 organ chlorine. Pesticides in horticulture sample by gas chromatography with electronic capture detection, J. Chromatogr. A. 849: 235-243. from developmental exposer to chloropyrifos., Brain. Res. Danso G, Drechsel P, Fialor SC (2002). Perception of organic agriculture by urban vegetable farmers and consumers in Ghana. Urban. Agriculture. Manag., 6: 23-24. EU Pesticides database. Available online with URL:http://ec.europa.eu/food/plant/pesticides/eupesticides database/public/?event=pesticide.residue. CurrentMRL&language=EN European Commission Health and Consumer Protection Directorate General. SANCO/12571/2013 (2013). Guidance document on analytical quality control and validation procedures for pesticide residues analysis in food and feed.,pp. 1-46. European Community (1990). EC Council Directive 90/642/EEC of 27 November 1990 on the fixing of maximum levels for pesticide residues in and on fruit and vegetables. In: Official J. the European Communities, vol. L350.European Community, Brussels, 71. European Council Directives 76/895/EEC, 86/362/EEC, 86/363/EEC and 90/642/EEC. Hela DG, Lambropoulou DA, Konstantinou I K, Albains TA (2005). Environmental monitoring and ecologicalrisk assessment for pesticide contamination and effects in Lake Pamvotis, Northwestern Greece. Environ. Toxicol. Chem., 24: 1548-1556. Jayashree R, and Vasudavan N (2007). Persistance and distribution of endosulfan under field condition. Environ. Monit assess. 131: 475-87. Johnson DE, Seidler FJ, Slotkin TA (1998). Early Biochemical detection of detailed neurotoxicity resulting

Jagadish et al. 106 Bull. 45: 143-146. Kumari B, Kumar R, Madan VK, Singh R, Singh J, Kathpal TS (2003) Environment. Assess. 3: 311-318. Kumari B, Madan VK, Kumar R, Kathpal TS (2002). Monitoring of seasonal vegetables for pesticide residues. Environmental. Monitoring and Assessment. 74 (3): 263-270. Nolan RJ, Rick DL, Freshoar NL, Saunders JH (1984). Chloropyriphos;pharmacokinetics in human volunteers. Toxicol.Appl.Pharmacol., 73: 8-10. Ntow WJ, Gijzen HJ, Kelderman P, Drechsel P (2006). Farmer perceptions and pesticide use practices in vegetable production in Ghana. Pest. Management. Science. 62 (4): 356-365. Singh SP, Kumar KS, Tanwar R.S. (2004): Dissipation and decontamination of cypermethrin and fluvalinate residues in okra. Pestic. Res. J., 16: (2) 65-67. Solomon G, Ogunseitan OA, Kirsch J (2000). Pesticides and Human Health: A Resource for Health Care Professionals. Physicians for Social Responsibility, Los Angeles and Californians for Pesticide Reform, San Francisco. 60. Tomer N (2013). Determination of Chlorinated Pesticide in Vegetables, Cereals and Pulses by Gas Chromatography in East National Capital Region, Delhi, India Res. J. Agric. Forestry Sci., 1: 27-28. Torres CM, Pico Y, Manes, J (1996). Determination of pesticide residues in fruit and vegetables. J. Chromatogr. A 754: 301 331. Webley DJ (1994). Grain protectants and pesticide residues. Proceedings of the 6th International Working Conference on Stored-product Protection 2: 857-862. Wilsont PC, Foos JF (2006). Survey of carbamate and organophosphorous pesticide export from a south Florida (USA) agricultural watershed: Implications of sampling Frequency on ecological risk estimation. Environ. Toxicol. Chem., 25: 2847-2852.